NOTES
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of North Dakota on 12/21/14 For personal use only.
Exopolysaccharide depolymerases induced by Rhizobium bacteriophages' YVONNEM. BARNETA N D BEVERLEY HUMPHREY Scllool of Microbiology, Uni~~ersity of New Sotit11 Woles, Kensit~gtoti,New Solit11Wnles 2033, Alrslrcllicr Accepted June 16, 1975
B A R N E T , Y . M., and B. H U M P H H ~ 1975. Y . Exopolysaccharide depolymerases induced by R l ~ i zobilrtn bacteriophages. Can. J . Microbiol. 21: 1647-1650. Enzymes induced by two Rhiiobi~itn1riJolii bacteriophages caused depolymerization of strains tested, but did not, in exopolysaccharides from most R . rrifolii and R . I~grit~~inosctrrrtt~ general, attack the exopolysaccharides of R . ,n~liloti,the slow-growing I-hizobia, or Agrohncr'rilrm. Ca2+ and (or) Mg2+ were required for enzyme activity. In all strains tested, depolymerization of exopolysaccharide occurred when there was successful phage infection, but depolymerization also occurred with exopolysaccharides from nonsusceptible strains. B A R N E T ,Y . M., et B. H U M P H R E Y 1975. . Exopolysaccharide depolymerases induced by RlrizoOilrtn bacteriophages. Can. J . Microbiol. 21: 1647-1650. Des enzymes qui sont induites pardeux bacteriophages, associes hRliizohirrtn trifolii, peuvent depolymeriser les exopolysaccharides de la majorit6 des souches de R . trifolii et de R . legtrtnitlosctr~rtn.En general, ces enzymes ne peuvent pas attaquer les exopolysaccharides de R. tnelilori, des rhizobies a croissance lente, ou d'Agrobnct~riion.Le Ca2+ et (ou) le Mg2+ sont nkcessaires pour I'activite enzymatique. La dCpolymerisation de I'exopolysaccharide sernble necessaire, mais pas suffisante, pour le processus d'infection.
Depolymerases of bacterial extracellular slime or capsules have been induced by phages attacking members of the Enterobacteriaceae (Adams and Park 1956; Stirm 1968; Stirm et al. 1971; Sutherland 1967; Sutherland and Wilkinson 1965; Watson 1966), Azotobacter vinlnndii (Eklund and Wyss 1962), Alcaligenes faecaiis (Mare and Smit 1969), and Pseudon7onns aeruginosa (Bartell and Orr 1969). These phages commonly produce plaques surrounded by a "halo" of incomplete clearing caused by the diffusion of a depolymerase beyond the area of phage-lytic activity. Several phages attacking Rhizobiurn trifolii were observed to form plaques of this type (Barnet 1972). This note presents preliminary results of a search for phage-induced enzymes able to produce depolymerization of the exopolysaccharides of Rhizobizrm. Phage CT3, a contractile-tailed phage with a flexible baseplate bearing a number of spikes, and phage N T l , a noncontractile phage with n o observable tail appendages (Barnet 1972) were chosen for this investigation. Both formed clear plaques surrounded by a turbid halo. The host 'Received December 12, 1974.
range of phage CT3 was wide relative to that of NTl. Using methods and media described earlier (Barnet and Vincent 1970), phage CT3 was grown and assayed on R. trifolii SU91 and phage NT1 on R. trifoiii SU297/3 I. Bacteria for enzyme and exopolysaccharide production were grown in yeast mannitol broth (Vincent 1970). For enzyme production, logarithmic phase broth cultures of the host strain were inoculated with phage to give a final multiplicity of infection of l o F 2 . After 2 days of incubation at 26 "C, the bacterial cells were removed by centrifugation, and the supernate was concentrated 10-fold by placing it in a dialysis bag in contact with Carbowax 20 M (polyethylene glycol, mol. wt. 20 000, Union Carbide Corporation). A further 30- t o 50-fold enzyme concentration was effected by ammonium sulfate precipitation at 75% saturation. The precipitates were dissolved in 0.02 M phosphate buffer, pH 7.2, and dialyzed against the same buffer overnight at 4 "C. The preparation was used without further treatment as a crude enzyme solution. Exopolysaccharides were precipitated from the supernate of a 3- t o 7-day broth culture by the
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of North Dakota on 12/21/14 For personal use only.
1648
C A N . J. MICROBIOIL. VOL. 21, 1975
addition of 3 volumes of ethanol. Some samples of exopolysaccharide were further purified by Sevag deproteinization and then by two further precipitations in alcohol (Humphrey and Vincent 1959). For routine screening tests of enzyme activity, the exopolysaccharides were dissolved in 0.02 M phosphate buffer, pH 7.2, to produce a solution (about 20 mg/ml) which could be pipetted but was sufficiently viscous to adhere to the bottom of an inverted test tube (10 x 100 mm). Exopolysaccharide solution (0.1 ml) was mixed with either enzyme preparation (0.05 ml) or buffer (0.05 ml), and the tubes were sealed and incubated at 26°C. Tubes were examined for decreases in viscosity after 24 and 48 h. A test was scored as positive if, upon inversion of the two test tubes, the enzyme-treated sample flowed down the side of the tube while the buffer-control preparation remained adherent to the base. Supernates of cultures infected with phages CT3 and NTI contained a product which depolymerized the exopolysaccharide of their host rhizobial strains, R. trijiolii SU9 1 and SU297/3 1, respectively. No activity could be demonstrated in concentrates from broken, uninfected cells. It was therefore concluded that these phage:host systems produce a polysaccharide depolymerase. We have no evidence yet to indicate whether the enzyme is under the control of the bacterial genome and induced by phage growth or whether it is a de rzoljo phage protein, although by analogy with other systems, the latter seems more probable (Bartell and Orr 1969; Bessler et al. 1973; Stirm et al. 1971 ; Sutherland and Wilkinson 1965). Divalent cations were necessary for enzyme activity. For consistent results, 0.05 m M Ca2+ and 0.05 m M Mg2+ were added to the phosphate buffer used in preparing enzyme and substrate. The exopolysaccharides from some strains of R. trifolii which had previously been unaffected by the depolymerase showed a rapid and reproducible decrease in viscosity after addition of divalent cations. Conversely, enzyme activity was abolished when MI10 ethylenediaminetetraacetic acid (EDTA) was added to the buffer in which the gum was dissolved. The tube technique for testing viscosity changes is, at best, only semiquantitative. Use of a viscometer is both more precise and more sensitive. To define the reaction parameters, viscometry was used for one experiment. A crude
enzyme preparation containing 2.6 mg protein/ ml (estimated by the ultraviolet absorption method of Warburg and Christian as quoted by Layne 1957) was diluted 1/50 by mixing with a solution of purified exopolysaccharide (1.5 mg/ ml) incubated at 30 "C, and the viscosity changes were followed using an Ostwald viscometer (Fig. 1). A progress curve typical of that commonly found in enzyme reactions was obtained. The reaction velocity was about linear until the point of 30% viscosity decrease. Thereafter it decreased with time. The sensitivity of this method is much greater than with the tube technique, where activity was not demonstrable with greater than fourfold dilution of the crude enzyme. However, since viscometry is not convenient for screening tests, it was not used subsequently in this work. Enzyme preparations from both phages were tested against exopolysaccharides from a number of Rllizobium and Agrobacterium strains (Table 1). Most exopolysaccharides from R . trifolii and R . legumirzosarum were liquefied by the enzymes, although a few of these reacted with
1 0
2 0
3 0 Tnme
4 0 Iminl
50
60
FIG.1. The effect of depolymerase from phage CT3 infection of R. trifolii SU91, on purified exopolysaccharide from the homologous strain. Purified exopolysaccharide, diluted in 0.02 M phosphate buffer ( p H 7.2) to give a final concentration of 1.5 rng/ml (relative viscosity 24) was mixed with crude enzyme (final concentration 1/50 of original preparation or 0.05 mg proteinjml). The mixture was incubated at 30°C and the relative viscosity determined at intervals with an Ostwald viscometer.
NOTES
TABLE 1
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Proportion of tested strains of Rhizobiu~nand Agrobacferium showing enzymic depolymerization of exopolysaccharide Enzyme source Source of exopolysaccharide
CT3 infecting SU91
NT1 infecting SU297131
R. frifolii R. legumirtosar~ml R. melilori R. japonicum R. lupini Lotus rhizobia Leucaena rhizobia Agrobacferiu~nspp.
only one of the enzyme systems. Exopolysaccharides from some slow-growing R. japonicunl strains were occasionally attacked, but showed a slower and less-marked decrease in viscosity than did the R. trifolii / R. leguminosarutn preparations. Exopolysaccharides from lupin. Lotus, and Leucaena rhizobia, the agrobacteria, and all but one R. meliloti strain failed to liquefy. The main differences which Zevenhuisen (1973) has listed between linkages detected in the R. trifolii / R. leglrminosarum group and the R. meliloti / Agrobacterium group are the occurrence of (1 4 4) linked D-glucuronic acid and of (1 4 4, 1 -t 6) branching residues of galactose exclusively in the former group. There is no firm correlation between the presence of glucuronic acid in the exopolysaccharide and susceptibility to liquefaction by the enzymes. Some of the exopolysaccharides from slow-growing rhizobia which failed to depolymerize are reported to contain glucuronic acid in significant amounts (20%; Bailey et al. 1971), but the linkages are not described. On the other hand, the one R. meliloti strain of the six tested which was liquefied by the enzymes showed the usual lack of glucuronic acid exhibited by these strains (2%; Humphrey and Vincent 1959; Bjorndal et 01. 1971; Zevenhuisen 1971). Paper chromatography of exopolysaccharides liquefied by the enzymes (solvent, butanol: pyridine : benzene :water = 5 : 3 : 3 : 1 ; spray reagent, 3% p-anisidine HCl in moist butanol) showed that no monosaccharides had been released by enzymic action. As has been found by other workers, there was
1649
no correlation between phage-host range (measured by plaque formation) and susceptibility of the exopolysaccharide to depolymerization (Adams and Park 1956; Sutherland and Wilkinson 1965). Enzymes from both phage-host systems could produce viscosity changes in exopolysaccharides from many strains on which they were unable to form plaques. This is to be expected since depolymerization of viscous exopolysaccharide to allow ready access of phage to the cell wall would be only the first in many steps required for phage infection and growth. However, in none of the strains tested was phage able to effect a productive infection without a parallel susceptibility of the exopolysaccharide t o the phage-induced depolymerase~ Acknowledgments Our thanks are due to Miss Helen Grigg for expert technical assistance, and to Professor J. M. Vincent for helpful advice. The work was supported by the Rural Credits Division of the Reserve Bank of Australia. ADAMS,M. H.. and B. H. P A R K 1956. . An enzyme produced by a phage-host cell system. 11. The properties of the polysaccharidedepolymerase. Virology,2: 719-736. B~ILEY R ., W . , R. M. GREENWOOD, and A. C R A I G 1971. . Extracellular polysaccharides of Rlzi~obirr1r7strains associated with Lorrrs species. J. Gen. Microbiol. 65: 315-324. BARNET. Y. M. 1972. BacteriophagesofRIli;ohirln7 ~rjfblii. I. Morphology and host range. J. Gen. Virol. 15: 1-15. BARNET,Y . M., and J . M. VINCENT.1970. Lysogenic conver-sion of Rl~izohirrii?Ir(fu1ii.J . Gen. Microbiol. 61: 3 19-325. B A R I X L LP. , F . , and '1.. E. O R R . 1969. Distinct slime polysaccharide depolymerases of bacteriophage-infecled Pserrdo~no~lcrs cierrrgi~zosn: evidence of close association with structured bacteriophage particle. J . Virol. 4: 580-584. BESSLER,W., E. FREUND-MOLBERT. H. K N U F E R M A N N . C. R U D O L P HH. , T H U R O Wand . S. S T I R M1973. . A bacteriophage-induced depolymerase active on Klehsielln K l l capsular polysaccharide. Virology, 56: 134-151. FA . HRA~US, BJORNDAL. H., C. E R B I N GB., L I N D B E R G , G and H. IJUNCGREN,1971. Studies on an extracellular polysaccharide from Rlrizobirr~nmelilori. Acta Chem. Scand. 25: 1281-1286. E C K L U N DC., , and 0. Wuss. 1962. Enzyme associated with bacteriophage infection. J . Bacterial. 84: 12091215. HUMPHREY. B. A , , and J . M. V I N C E N T1959. . Extracellular polysaccharidesofRlrizobirrn~. J. Gen. Microbiol. 21: 477-484. L A Y N E E. , 1957. Spectrophotometric and turbidimetric methods for measuring protein. Methods Enzymol. 3: 447 -454. MARC, I. 1.. and J . A. S ~ I I T .1969. A capsule-
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of North Dakota on 12/21/14 For personal use only.
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depolymerizing enzyme from Alccrliget~esfaecnlis infected with bacteriophage A6. J. Gen. Virol. 5: 551-552. S T I R MS. , 1968. Eschericl~incoli K bacteriophages. I. Isolation and introductory characterization of five Esckcrichicr coli K bacteriophages. J. Virol. 2: 1 107-1 1 14. STIRM. S., W. BESSLER, F. F E H M E L .E. F R E U N D MOLRERT, and H. T H U R O W1971. . Isolation of spikeformed particles from bacteriophage lysates. Virology, 45: 303-308. S U . ~ H E R L A N1.DW. . 1967. Phage-induced fucosidases hydrolysing the exopolysaccharide of Klebsiella rrerogetles Type 54 A3(Sl). Biochem. J. 104: 278-285. SUTHERLAND I. , W., and J . F. W I L K I N S O N 1965. . Depolymerases for bacterial exopolysaccharides obtained
VOL. 21,
1975
from phage-infected bacteria. J. Gen. Miclobiol. 39: 373-383. VINCENT, J. M . 1970. A manual for the practical study of the root-nodule bacteria. IBP Handbook No. 15. Blackwell, Oxford and Edinburgh, p. 3. WATSON,K. C. 1966. The effect of polysaccharide depolymerizing enzyme in gel diffusion and haemagglutination studies. Immunology, 10: 121-126. Z E V E N H U I S EL. N ,P. T. M. 1971. Chemicalcomposition of the exopolysaccharides of R h i z o b i ~ r ~and n Agrobcrctc,rirrln. J. Gen. Microbiol. 68: 239-243. -1973. Methylation analysis of acidic exopolysaccharides of R l ~ i z o b i r l tand ~ AgJ'obrrcterili~n.Carbohydr. Res. 26: 409419.
Concanavalin A as a selective agent in tissue culture for temperature-sensitive hamster cell lines' J I M A. WRIGHT D~,pcirrr~retlt of'Microl>iology,Utri~~er.sity c~fMotlitobo,Wintlipeg, Mcrtlitobo R3TZN2 Accepted June 16, 1975
W H I G H J~. , A. 1975. Concanavalin A a s a selective agent in tissue culture for temperaturesensitive hamster cell lines. Can. J. Microbiol. 21: 1650-1654. A novel method for- isolating Chinese hamster cell cultures with temperature-sensitive growth properties is described. Concanavalin A can be used as aselective agent in tissue culture to isolate lectin-resistant cell lines which exhibit colony-forming abilities at the nonpermissive temperatllre relative to the permissive temperature as low a s 10-4 to A general correlation exists between resistance to the lectin and temperature sensitivity. W R I G H TJ. . A. 1975. Concanavalin A as a selective agent in tissue culture for temperaturesensitive hamstercell lines. Can. J . Microbiol. 21: 1650-1654. Une nouvelle methode est dkcrite pour isoler des cultures de cellules de hamsters chinois dont la croissance est thermosensible. L'utilisation de concanavaline A comme agent selecteur dans des cultures de tissus permet d'isoler des lignees cellulaires rbsistantes aux lectines qui ont cette a p t i t ~ ~ ddee former des colonies dans I'ordre de B B des temperatures non-permises comparativement i des temperatures permises. Une correlation generale existe entre la resistance aux lectines e t la sensibilite B la temperature. [Traduit par le journal]
Studies on microorganisms with conditional lethal mutations have considerably improved our understanding of regulatory systems in the procaryotic cell. Therefore, much interest has developed in obtaining proccdures for isolating and studying conditional lethal temperaturesensitive (fs) mutations of somatic animal cells in tissue culture. In most cases these isolation procedures were designed t o select for noncycling cells at the nonpermissive temperature by adding drugs t o the growth medium whose action was directed towards the S phase of the cell cycle (5, 'Received February 25, 1975.
6, 8, 9, 10). These methods have proved t o be very useful a n d some of the mutants are being subjected t o intensive study. The purpose of this communication is to report a novel method for isolating Chinese hamster ovary (CHO) cell lines with obvious ts growth properties. This new procedure uses concanavalin A (con A) a s a selective agent in tissue culture to obtain lectin-resistant cell lines which exhibit colony-forming efficiencies at 39 "C relative to those at 34 "C as low as to 10-6 CHO cells (4) were routinely grown a s monolayer cultures in a CO, a n d humidity controlled
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Exopolysaccharide depolymerases induced by Rhizobium bacteriophages' YVONNEM. BARNETA N D BEVERLEY HUMPHREY Scllool of Microbiology, Uni~~ersity of New Sotit11 Woles, Kensit~gtoti,New Solit11Wnles 2033, Alrslrcllicr Accepted June 16, 1975
B A R N E T , Y . M., and B. H U M P H H ~ 1975. Y . Exopolysaccharide depolymerases induced by R l ~ i zobilrtn bacteriophages. Can. J . Microbiol. 21: 1647-1650. Enzymes induced by two Rhiiobi~itn1riJolii bacteriophages caused depolymerization of strains tested, but did not, in exopolysaccharides from most R . rrifolii and R . I~grit~~inosctrrrtt~ general, attack the exopolysaccharides of R . ,n~liloti,the slow-growing I-hizobia, or Agrohncr'rilrm. Ca2+ and (or) Mg2+ were required for enzyme activity. In all strains tested, depolymerization of exopolysaccharide occurred when there was successful phage infection, but depolymerization also occurred with exopolysaccharides from nonsusceptible strains. B A R N E T ,Y . M., et B. H U M P H R E Y 1975. . Exopolysaccharide depolymerases induced by RlrizoOilrtn bacteriophages. Can. J . Microbiol. 21: 1647-1650. Des enzymes qui sont induites pardeux bacteriophages, associes hRliizohirrtn trifolii, peuvent depolymeriser les exopolysaccharides de la majorit6 des souches de R . trifolii et de R . legtrtnitlosctr~rtn.En general, ces enzymes ne peuvent pas attaquer les exopolysaccharides de R. tnelilori, des rhizobies a croissance lente, ou d'Agrobnct~riion.Le Ca2+ et (ou) le Mg2+ sont nkcessaires pour I'activite enzymatique. La dCpolymerisation de I'exopolysaccharide sernble necessaire, mais pas suffisante, pour le processus d'infection.
Depolymerases of bacterial extracellular slime or capsules have been induced by phages attacking members of the Enterobacteriaceae (Adams and Park 1956; Stirm 1968; Stirm et al. 1971; Sutherland 1967; Sutherland and Wilkinson 1965; Watson 1966), Azotobacter vinlnndii (Eklund and Wyss 1962), Alcaligenes faecaiis (Mare and Smit 1969), and Pseudon7onns aeruginosa (Bartell and Orr 1969). These phages commonly produce plaques surrounded by a "halo" of incomplete clearing caused by the diffusion of a depolymerase beyond the area of phage-lytic activity. Several phages attacking Rhizobiurn trifolii were observed to form plaques of this type (Barnet 1972). This note presents preliminary results of a search for phage-induced enzymes able to produce depolymerization of the exopolysaccharides of Rhizobizrm. Phage CT3, a contractile-tailed phage with a flexible baseplate bearing a number of spikes, and phage N T l , a noncontractile phage with n o observable tail appendages (Barnet 1972) were chosen for this investigation. Both formed clear plaques surrounded by a turbid halo. The host 'Received December 12, 1974.
range of phage CT3 was wide relative to that of NTl. Using methods and media described earlier (Barnet and Vincent 1970), phage CT3 was grown and assayed on R. trifolii SU91 and phage NT1 on R. trifoiii SU297/3 I. Bacteria for enzyme and exopolysaccharide production were grown in yeast mannitol broth (Vincent 1970). For enzyme production, logarithmic phase broth cultures of the host strain were inoculated with phage to give a final multiplicity of infection of l o F 2 . After 2 days of incubation at 26 "C, the bacterial cells were removed by centrifugation, and the supernate was concentrated 10-fold by placing it in a dialysis bag in contact with Carbowax 20 M (polyethylene glycol, mol. wt. 20 000, Union Carbide Corporation). A further 30- t o 50-fold enzyme concentration was effected by ammonium sulfate precipitation at 75% saturation. The precipitates were dissolved in 0.02 M phosphate buffer, pH 7.2, and dialyzed against the same buffer overnight at 4 "C. The preparation was used without further treatment as a crude enzyme solution. Exopolysaccharides were precipitated from the supernate of a 3- t o 7-day broth culture by the
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of North Dakota on 12/21/14 For personal use only.
1648
C A N . J. MICROBIOIL. VOL. 21, 1975
addition of 3 volumes of ethanol. Some samples of exopolysaccharide were further purified by Sevag deproteinization and then by two further precipitations in alcohol (Humphrey and Vincent 1959). For routine screening tests of enzyme activity, the exopolysaccharides were dissolved in 0.02 M phosphate buffer, pH 7.2, to produce a solution (about 20 mg/ml) which could be pipetted but was sufficiently viscous to adhere to the bottom of an inverted test tube (10 x 100 mm). Exopolysaccharide solution (0.1 ml) was mixed with either enzyme preparation (0.05 ml) or buffer (0.05 ml), and the tubes were sealed and incubated at 26°C. Tubes were examined for decreases in viscosity after 24 and 48 h. A test was scored as positive if, upon inversion of the two test tubes, the enzyme-treated sample flowed down the side of the tube while the buffer-control preparation remained adherent to the base. Supernates of cultures infected with phages CT3 and NTI contained a product which depolymerized the exopolysaccharide of their host rhizobial strains, R. trijiolii SU9 1 and SU297/3 1, respectively. No activity could be demonstrated in concentrates from broken, uninfected cells. It was therefore concluded that these phage:host systems produce a polysaccharide depolymerase. We have no evidence yet to indicate whether the enzyme is under the control of the bacterial genome and induced by phage growth or whether it is a de rzoljo phage protein, although by analogy with other systems, the latter seems more probable (Bartell and Orr 1969; Bessler et al. 1973; Stirm et al. 1971 ; Sutherland and Wilkinson 1965). Divalent cations were necessary for enzyme activity. For consistent results, 0.05 m M Ca2+ and 0.05 m M Mg2+ were added to the phosphate buffer used in preparing enzyme and substrate. The exopolysaccharides from some strains of R. trifolii which had previously been unaffected by the depolymerase showed a rapid and reproducible decrease in viscosity after addition of divalent cations. Conversely, enzyme activity was abolished when MI10 ethylenediaminetetraacetic acid (EDTA) was added to the buffer in which the gum was dissolved. The tube technique for testing viscosity changes is, at best, only semiquantitative. Use of a viscometer is both more precise and more sensitive. To define the reaction parameters, viscometry was used for one experiment. A crude
enzyme preparation containing 2.6 mg protein/ ml (estimated by the ultraviolet absorption method of Warburg and Christian as quoted by Layne 1957) was diluted 1/50 by mixing with a solution of purified exopolysaccharide (1.5 mg/ ml) incubated at 30 "C, and the viscosity changes were followed using an Ostwald viscometer (Fig. 1). A progress curve typical of that commonly found in enzyme reactions was obtained. The reaction velocity was about linear until the point of 30% viscosity decrease. Thereafter it decreased with time. The sensitivity of this method is much greater than with the tube technique, where activity was not demonstrable with greater than fourfold dilution of the crude enzyme. However, since viscometry is not convenient for screening tests, it was not used subsequently in this work. Enzyme preparations from both phages were tested against exopolysaccharides from a number of Rllizobium and Agrobacterium strains (Table 1). Most exopolysaccharides from R . trifolii and R . legumirzosarum were liquefied by the enzymes, although a few of these reacted with
1 0
2 0
3 0 Tnme
4 0 Iminl
50
60
FIG.1. The effect of depolymerase from phage CT3 infection of R. trifolii SU91, on purified exopolysaccharide from the homologous strain. Purified exopolysaccharide, diluted in 0.02 M phosphate buffer ( p H 7.2) to give a final concentration of 1.5 rng/ml (relative viscosity 24) was mixed with crude enzyme (final concentration 1/50 of original preparation or 0.05 mg proteinjml). The mixture was incubated at 30°C and the relative viscosity determined at intervals with an Ostwald viscometer.
NOTES
TABLE 1
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Proportion of tested strains of Rhizobiu~nand Agrobacferium showing enzymic depolymerization of exopolysaccharide Enzyme source Source of exopolysaccharide
CT3 infecting SU91
NT1 infecting SU297131
R. frifolii R. legumirtosar~ml R. melilori R. japonicum R. lupini Lotus rhizobia Leucaena rhizobia Agrobacferiu~nspp.
only one of the enzyme systems. Exopolysaccharides from some slow-growing R. japonicunl strains were occasionally attacked, but showed a slower and less-marked decrease in viscosity than did the R. trifolii / R. leguminosarutn preparations. Exopolysaccharides from lupin. Lotus, and Leucaena rhizobia, the agrobacteria, and all but one R. meliloti strain failed to liquefy. The main differences which Zevenhuisen (1973) has listed between linkages detected in the R. trifolii / R. leglrminosarum group and the R. meliloti / Agrobacterium group are the occurrence of (1 4 4) linked D-glucuronic acid and of (1 4 4, 1 -t 6) branching residues of galactose exclusively in the former group. There is no firm correlation between the presence of glucuronic acid in the exopolysaccharide and susceptibility to liquefaction by the enzymes. Some of the exopolysaccharides from slow-growing rhizobia which failed to depolymerize are reported to contain glucuronic acid in significant amounts (20%; Bailey et al. 1971), but the linkages are not described. On the other hand, the one R. meliloti strain of the six tested which was liquefied by the enzymes showed the usual lack of glucuronic acid exhibited by these strains (2%; Humphrey and Vincent 1959; Bjorndal et 01. 1971; Zevenhuisen 1971). Paper chromatography of exopolysaccharides liquefied by the enzymes (solvent, butanol: pyridine : benzene :water = 5 : 3 : 3 : 1 ; spray reagent, 3% p-anisidine HCl in moist butanol) showed that no monosaccharides had been released by enzymic action. As has been found by other workers, there was
1649
no correlation between phage-host range (measured by plaque formation) and susceptibility of the exopolysaccharide to depolymerization (Adams and Park 1956; Sutherland and Wilkinson 1965). Enzymes from both phage-host systems could produce viscosity changes in exopolysaccharides from many strains on which they were unable to form plaques. This is to be expected since depolymerization of viscous exopolysaccharide to allow ready access of phage to the cell wall would be only the first in many steps required for phage infection and growth. However, in none of the strains tested was phage able to effect a productive infection without a parallel susceptibility of the exopolysaccharide t o the phage-induced depolymerase~ Acknowledgments Our thanks are due to Miss Helen Grigg for expert technical assistance, and to Professor J. M. Vincent for helpful advice. The work was supported by the Rural Credits Division of the Reserve Bank of Australia. ADAMS,M. H.. and B. H. P A R K 1956. . An enzyme produced by a phage-host cell system. 11. The properties of the polysaccharidedepolymerase. Virology,2: 719-736. B~ILEY R ., W . , R. M. GREENWOOD, and A. C R A I G 1971. . Extracellular polysaccharides of Rlzi~obirr1r7strains associated with Lorrrs species. J. Gen. Microbiol. 65: 315-324. BARNET. Y. M. 1972. BacteriophagesofRIli;ohirln7 ~rjfblii. I. Morphology and host range. J. Gen. Virol. 15: 1-15. BARNET,Y . M., and J . M. VINCENT.1970. Lysogenic conver-sion of Rl~izohirrii?Ir(fu1ii.J . Gen. Microbiol. 61: 3 19-325. B A R I X L LP. , F . , and '1.. E. O R R . 1969. Distinct slime polysaccharide depolymerases of bacteriophage-infecled Pserrdo~no~lcrs cierrrgi~zosn: evidence of close association with structured bacteriophage particle. J . Virol. 4: 580-584. BESSLER,W., E. FREUND-MOLBERT. H. K N U F E R M A N N . C. R U D O L P HH. , T H U R O Wand . S. S T I R M1973. . A bacteriophage-induced depolymerase active on Klehsielln K l l capsular polysaccharide. Virology, 56: 134-151. FA . HRA~US, BJORNDAL. H., C. E R B I N GB., L I N D B E R G , G and H. IJUNCGREN,1971. Studies on an extracellular polysaccharide from Rlrizobirr~nmelilori. Acta Chem. Scand. 25: 1281-1286. E C K L U N DC., , and 0. Wuss. 1962. Enzyme associated with bacteriophage infection. J . Bacterial. 84: 12091215. HUMPHREY. B. A , , and J . M. V I N C E N T1959. . Extracellular polysaccharidesofRlrizobirrn~. J. Gen. Microbiol. 21: 477-484. L A Y N E E. , 1957. Spectrophotometric and turbidimetric methods for measuring protein. Methods Enzymol. 3: 447 -454. MARC, I. 1.. and J . A. S ~ I I T .1969. A capsule-
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of North Dakota on 12/21/14 For personal use only.
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depolymerizing enzyme from Alccrliget~esfaecnlis infected with bacteriophage A6. J. Gen. Virol. 5: 551-552. S T I R MS. , 1968. Eschericl~incoli K bacteriophages. I. Isolation and introductory characterization of five Esckcrichicr coli K bacteriophages. J. Virol. 2: 1 107-1 1 14. STIRM. S., W. BESSLER, F. F E H M E L .E. F R E U N D MOLRERT, and H. T H U R O W1971. . Isolation of spikeformed particles from bacteriophage lysates. Virology, 45: 303-308. S U . ~ H E R L A N1.DW. . 1967. Phage-induced fucosidases hydrolysing the exopolysaccharide of Klebsiella rrerogetles Type 54 A3(Sl). Biochem. J. 104: 278-285. SUTHERLAND I. , W., and J . F. W I L K I N S O N 1965. . Depolymerases for bacterial exopolysaccharides obtained
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from phage-infected bacteria. J. Gen. Miclobiol. 39: 373-383. VINCENT, J. M . 1970. A manual for the practical study of the root-nodule bacteria. IBP Handbook No. 15. Blackwell, Oxford and Edinburgh, p. 3. WATSON,K. C. 1966. The effect of polysaccharide depolymerizing enzyme in gel diffusion and haemagglutination studies. Immunology, 10: 121-126. Z E V E N H U I S EL. N ,P. T. M. 1971. Chemicalcomposition of the exopolysaccharides of R h i z o b i ~ r ~and n Agrobcrctc,rirrln. J. Gen. Microbiol. 68: 239-243. -1973. Methylation analysis of acidic exopolysaccharides of R l ~ i z o b i r l tand ~ AgJ'obrrcterili~n.Carbohydr. Res. 26: 409419.
Concanavalin A as a selective agent in tissue culture for temperature-sensitive hamster cell lines' J I M A. WRIGHT D~,pcirrr~retlt of'Microl>iology,Utri~~er.sity c~fMotlitobo,Wintlipeg, Mcrtlitobo R3TZN2 Accepted June 16, 1975
W H I G H J~. , A. 1975. Concanavalin A a s a selective agent in tissue culture for temperaturesensitive hamster cell lines. Can. J. Microbiol. 21: 1650-1654. A novel method for- isolating Chinese hamster cell cultures with temperature-sensitive growth properties is described. Concanavalin A can be used as aselective agent in tissue culture to isolate lectin-resistant cell lines which exhibit colony-forming abilities at the nonpermissive temperatllre relative to the permissive temperature as low a s 10-4 to A general correlation exists between resistance to the lectin and temperature sensitivity. W R I G H TJ. . A. 1975. Concanavalin A as a selective agent in tissue culture for temperaturesensitive hamstercell lines. Can. J . Microbiol. 21: 1650-1654. Une nouvelle methode est dkcrite pour isoler des cultures de cellules de hamsters chinois dont la croissance est thermosensible. L'utilisation de concanavaline A comme agent selecteur dans des cultures de tissus permet d'isoler des lignees cellulaires rbsistantes aux lectines qui ont cette a p t i t ~ ~ ddee former des colonies dans I'ordre de B B des temperatures non-permises comparativement i des temperatures permises. Une correlation generale existe entre la resistance aux lectines e t la sensibilite B la temperature. [Traduit par le journal]
Studies on microorganisms with conditional lethal mutations have considerably improved our understanding of regulatory systems in the procaryotic cell. Therefore, much interest has developed in obtaining proccdures for isolating and studying conditional lethal temperaturesensitive (fs) mutations of somatic animal cells in tissue culture. In most cases these isolation procedures were designed t o select for noncycling cells at the nonpermissive temperature by adding drugs t o the growth medium whose action was directed towards the S phase of the cell cycle (5, 'Received February 25, 1975.
6, 8, 9, 10). These methods have proved t o be very useful a n d some of the mutants are being subjected t o intensive study. The purpose of this communication is to report a novel method for isolating Chinese hamster ovary (CHO) cell lines with obvious ts growth properties. This new procedure uses concanavalin A (con A) a s a selective agent in tissue culture to obtain lectin-resistant cell lines which exhibit colony-forming efficiencies at 39 "C relative to those at 34 "C as low as to 10-6 CHO cells (4) were routinely grown a s monolayer cultures in a CO, a n d humidity controlled
